1. Field of the Invention
This invention relates to mutant purine nucleoside phosphorylase enzymes having different activity than the non-mutant purine nucleoside phosphorylase enzyme, and to substrates for these mutant enzymes. In particular the invention relates to a mutant M65V having greater activity than the wild-type enzyme in cleaving specific substrates.
2. Description of the Related Art
A prodrug activation strategy for selectively impairing tumor cells involves the expression of a gene encoding an exogenous enzyme in the tumor cells and administration of a substrate for that enzyme. The enzyme acts on the substrate to generate a substance toxic to the targeted tumor cells. This technique has advantages over the expression of directly toxic genes, such as ricin, diphtheria toxin, or pseudomonas exotoxin. These advantages include the capability to (1) titrate cell impairment, (2) optimize therapeutic index by adjusting either levels of prodrug or of recombinant enzyme expression, and (3) interrupt toxicity by omitting administration of the prodrug. In addition, this technique uses prodrugs found to have different effects on different cell types, allowing treatment to be adjusted according to a specific disease state.
Enzymes useful in a prodrug activation approach have been described and include enzymes such as thymidine kinase, cytosine deaminase and purine nucleoside phosphorylase, as described in U.S. Pat. Nos. 5,338,678, 5,552,311, 6,017,896 and 6,207,150. However, the effectiveness of tumor treatment using prodrug activation techniques may be limited in cases where side-effects of substrate administration are present. For example, the prodrug ganciclovir, often used in combination with thymidine kinase, can cause unwanted immunosuppressive effects. In the case of purine nucleoside phosphorylase therapy, undesirable side-effects may occur due to the activity of endogenous human and E. coli purine nucleoside phosphorylases present in human cells and in normal intestinal flora respectively.
Thus, there exists a need for a prodrug activation method for treating tumors that overcomes the problem of side effects.